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• • A—HUMAN NECESSITIES
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• • C—CHEMISTRY; METALLURGY
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings

Definitions

• the present invention relates to solid salt forms of a 3 -pyrrole substituted 2- indolinone compound, 5-[5-fluoro-2-oxo- 1 ,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4- dimethyl- lH-pyrrole-3-carboxy lie acid (2-pyrrolidin-l-yl-ethyl)-amide.
• the foregoing compounds modulate the activity of protein kinases ("PKs").
• PKs protein kinases
• the compounds of this invention are therefore useful in treating disorders related to abnormal PK activity.
• Pharmaceutical compositions comprising salts of this compound and methods of preparing them are disclosed.
• the present invention is also directed to polymorphs of the phosphate salt form of the amide.
• Polymorphism occurs when a compound crystallizes in a multiplicity of solid phases that differ in crystal packing. Numerous examples are cited in the standard references of solid state properties of pharmaceuticals, Byrn, S. R, Solid-State Chemistry of Drugs, New Your, Academic Press (1982); Kuhnert- Brandstatter, M., Thermomiscroscopy In The Analysis of Pharmaceuticals, New York, Pergamon Press (1971) and Haleblian, J. K. and McCrone, W. Pharmaceutical applications of polymorphism. J. Pharm. ScL, 58, 911 (1969). Byrn states that, in general, polymorphs exhibit different physical characteristics including solubility and physical and chemical stability.
• polymorphs may differ in ways that influence drug release, solid-state stability, and pharmaceutical manufacturing.
• the relative stability and the interconversions of polymorphs are particularly important to the selection of a marketed drug.
• a suitable polymorph may hinge upon the issue of physical stability.
• the selection of a marketed drug may depend upon the availability and selection of a suitable polymorph having desirable characteristics, such as excellent physical stability or the ability to be manufactured in large scale.
• the performance of the solid dosage form should not be limited by polymorphic transformations during the shelf life of the product. It is important to note that there is no reliable method to predict the observable crystal structures of a given drug or to predict the existence of polymorphs with desirable physical properties.
• PKs are enzymes that catalyze the phosphorylation of hydroxy groups on tyrosine, serine, and threonine residues of proteins.
• the consequences of this seemingly simple activity are staggering since virtually all aspects of cell life (e.g., cell growth, differentiation, and proliferation) in one way or another depend on PK activity.
• abnormal PK activity has been related to a host of disorders, ranging from relatively non-life threatening diseases such as psoriasis to extremely virulent diseases such as glioblastoma (brain cancer).
• Receptor tyrosine kinases are excellent candidates for molecular targeted therapy, because they play key roles in controlling cell proliferation and survival and are frequently dysregulated in a variety of malignancies.
• the mechanisms of dysregulation include overexpression (Her2/neu in breast cancer, epidermal growth factor receptor in non-small cell lung cancer), activating mutations (KIT in gastrointestinal stromal tumors, fms-related tyrosine kinase 3/Flk2 (FLT3) in acute myelogenous leukemia), and autocrine loops of activation (vascular endothelial growth factor/VEGF receptor (VEGF/VEGFR) in melanoma, platelet-derived growth factor/PDGF receptor (PDGF/PDGFR) in sarcoma).
• overexpression Her2/neu in breast cancer, epidermal growth factor receptor in non-small cell lung cancer
• KIT gastrointestinal stromal tumors
• FLT3 fms-related tyrosine kinase 3/Fl
• the RTKs and their ligands, VEGF, PDGF, and FGF mediate neovascularization, known as angiogenesis, in solid tumors. Consequently, by inhibiting the RTKs, the growth of new blood vessels into tumors may be inhibited.
• Antiangiogenesis agents a class of molecules that inhibits the growth of blood vessels into tumors, have much less toxicity to the body compared to conventional anti-cancer drugs.
• US patent 6,573,293, incorporated herein by reference discloses, among other compounds, 5-[5-fluoro-2-oxo-l,2-dihydro-indol-(3Z)-ylidenemethyl]- 2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2-pyrrolidin- l-yl-ethyl)-amide (hereinafter "Compound I"). It has the following structure:
• Compound I is a small molecule that exhibits PK modulating ability. The compound is therefore useful in treating disorders related to abnormal PK activity. It is an inhibitor of the RTKs, PDGFR, VEGFR, KIT, and FLT3. Compound I has been shown to inhibit KIT phosphorylation, arrest cell proliferation, and induce cell cycle arrest and apoptosis in malignant mast cell lines in vitro expressing various forms of mutant KIT. Compound I and related molecules are effective in preclinical models against tumor xenografts arising from cell lines of diverse human tumor origin.
• Compound I is useful for treating cancers in companion animals, mainly dogs, and is also useful for the treatment of, inter alia, cancer in humans.
• cancers include, but are not limited to, leukemia, brain cancer, non-small cell lung cancer, squamous cell carcinoma, astrocytoma, Kaposi's sarcoma, glioblastoma, lung cancer, bladder cancer, head and neck cancer, small-cell lung cancer, glioma, colorectal cancer, genitourinary cancer, and gastrointestinal stromal cancer.
• Compound I is useful for the treatment of diseases related to overexpression of mast cells, including but not limited to, mastocytosis in humans and mast cell tumors in dogs.
• the phosphate salt form with a molecular formula OfC 22 H 25 FN 4 O 2 -H 3 O 4 P is selected.
• the phosphate salt form with a melting point from about 285 to about 290° C is selected.
• Compound I phosphate has a structure of
• a second aspect of the invention is a pharmaceutical composition comprising the phosphate salt or the citrate salt of Compound I, or solvates or polymorphs thereof, and a pharmaceutically acceptable carrier or excipient.
• a third aspect of the invention is a method for the modulation of the catalytic activity of protein kinases comprising contacting said protein kinase with the phosphate or citrate salts of Compound I, or solvates or polymorphs thereof.
• the protein kinase may be selected from the group consisting of receptor tyrosine kinases, non-receptor protein tyrosine kinases, and serine/threonine protein kinases.
• a fifth aspect of the invention is a method of preparing phosphate salt crystals ofbase 5-[5-fluoro-2-oxo-l,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-lH- pyrrole-3-carboxylic acid (2-pyrrolidin-l-yl-ethyl)-amide which comprises introducing a stoichiometric amount of phosphoric acid to the base in a solution comprising a solvent or a mixture of solvents, forcing the phosphate salt in solution to crystallize, separating the phosphate salt crystals from the solvent solution, and drying the crystals.
• FIG. 4 TGA curves of solids from CH 2 Cl 2 (Form VI, immediately after precipitation), Hexane (Form VII, after standing overnight), and acetonitrile (Form VIII, after standing 3 days).
• Figure 5. Results of agarose gel electrophoresis of PCR products from MCTs evaluated in Example 7. Lanes 1-5 correspond to patients 1-5 in Table 8; Lanes 6-14 correspond to patients 6-14 in Table 8. Controls consisted of PCR products generated from the C2 canine mast cell line containing a 48-bp ITD (Lane 15) and from normal canine cerebellum (wild type; Lane 16).
• Figure 6. Reductions in MCT phosphorylated KIT and phosphorylated extracellular signal-regulated kinase (ERK) 1/2 after a single dose of Compound I phosphate
• contacting refers to bringing a compound of the present invention and a target PK together in such a manner that the compound can affect the catalytic activity of the PK, either directly, i.e., by interacting with the kinase itself, or indirectly, i.e., by interacting with another molecule on which the catalytic activity of the kinase is dependent.
• PK refers to receptor protein tyrosine kinase (RTKs), non-receptor or “cellular” tyrosine kinase (CTKs) and serine-threonine kinases (STKs).
• RTKs receptor protein tyrosine kinase
• CTKs non-receptor or “cellular” tyrosine kinase
• STKs serine-threonine kinases
• 'solvate' is used to describe a molecular complex comprising a compound of the invention and one or more pharmaceutically acceptable solvent molecules, for example, ethanol.
• solvent molecules for example, ethanol.
• 'hydrate' is employed when said solvent is water.
• Carriers and excipients for formulation of pharmaceutically acceptable compositions comprising Compound I are well known in the art and are disclosed, for example, in U.S. Patent No. 6,573,293, which is incorporated herein in its entirety. Methods of administration for such are also known in the art and also described, for example, in U.S. Patent No. 6,573,293. Similar methods could also be used to formulate and administer pharmaceutically acceptable compositions of the salts of Compound I, or the polymorphs of such salts, of this invention.
• Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably adjusted to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. Additionally, suspensions of the compounds of the present invention may be prepared in a lipophilic vehicle. Suitable lipophilic vehicles include fatty oils such as sesame oil, synthetic fatty acid esters, such as ethyl oleate and triglycerides, or materials such as liposomes. The compounds of the invention may be administered orally.
• the compounds of the invention may also be administered topically, (intra)dermally, or transdermally to the skin or mucosa.
• Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibres, bandages and microemulsions. Liposomes may also be used.
• Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol.
• Penetration enhancers may be incorporated - see, for example, J Pharm Sci, 88 (10), 955-958, by Finnin and Morgan (October 1999).
• Other means of topical administration include delivery by electroporation, iontophoresis, phonophoresis, sonophoresis and microneedle or needle-free (e.g. PowderjectTM, BiojectTM, etc.) injection
• the compounds of the present invention may be formulated for rectal administration, such as suppositories or retention enemas using, for example, conventional suppository bases such as cocoa butter or other glycerides.
• the compounds of the present invention may also exist in unsolvated and solvated forms.
• the embodiments of the present invention also contemplate a method for the modulation of the catalytic activity of a PK comprising contacting said PK with a one or more of the salts of Compound I or the polymorphs of such salts of the present invention.
• Such "contacting” can be accomplished “in vitro " i.e., in a test tube, a petri dish, or the like.
• contacting may involve only a compound and a PK of interest or it may involve whole cells. Cells may also be maintained or grown in cell culture dishes and contacted with a compound in that environment.
• the ability of a particular compound to affect a PK-related disorder i.e., the IC 50 of the compound, defined below, can be determined before use of the compounds is attempted in vivo with more complex living organisms.
• a PK-related disorder i.e., the IC 50 of the compound, defined below.
• Embodiments of the present invention contemplate a method for treating or preventing a protein kinase related disorder in an organism (e.g., a companion animal or a human) comprising administering a therapeutically effective amount of a pharmaceutical composition comprising one or more of the salts of Compound I or the polymorphs of such salts of the present invention and a pharmaceutically acceptable carrier or excipient to the organism.
• an organism e.g., a companion animal or a human
• a pharmaceutical composition comprising one or more of the salts of Compound I or the polymorphs of such salts of the present invention and a pharmaceutically acceptable carrier or excipient to the organism.
• the protein kinase related disorder is selected from the group consisting of a receptor tyrosine kinase related disorder, a non-receptor tyrosine kinase related disorder, and a serine-threonine kinase related disorder.
• the protein kinase related disorder is selected from the group consisting of an EGFR related disorder, a PDGFR related disorder, an IGFR related disorder, and a FLK related disorder.
• the receptor protein kinase whose catalytic activity is modulated by a compound of this invention is selected from the group consisting of EGF, HER2, HER3, HER4, IR, IGF- IR, IRR, PDGFR ⁇ , PDGFR ⁇ , CSFIR, C-Kit, C-fms, FIk- IR, Flk4, KDR/Flk-1, FIt-I, FGFR-IR, FGFR-2R, FGFR-3R and FGFR-4R.
• the cellular tyrosine kinase whose catalytic activity is modulated by a compound of this invention is selected from the group consisting of Src, Frk, Btk, Csk, AbI, ZAP70, Fes/Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck, BIk, Hck, Fgr and Yrk.
• the serine-threonine protein kinase whose catalytic activity is modulated by a compound of this invention is selected from the group consisting of CDK2 and Raf.
• the protein kinase related disorder is selected from the group consisting of squamous cell carcinoma, astrocytoma, Kaposi's sarcoma, glioblastoma, lung cancer, bladder cancer, head and neck cancer, melanoma, ovarian cancer, prostate cancer, breast cancer, small cell lung cancer, glioma, colorectal cancer, genitourinary cancer, gastrointestinal cancer, mastocytosis, and mast cell tumors.
• the protein kinase related disorder is selected from the group consisting of diabetes, an autoimmune disorder, a hyperproliferation disorder, restenosis, fibrosis, psoriasis, von Heppel-Lindau disease, osteoarthritis, rheumatoid arthritis, angiogenesis, an inflammatory disorder, an immunological disorder, and a cardiovascular disorder.
• the amount of the compound to be administered ranges from about 0.001 to about 100 mg per kg of body weight, such total dose being given at one time or in divided doses.
• the amount of a composition administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician or veterinarian, etc.
• the effective local concentration of the drug may not be related to plasma concentration and other procedures known in the art may be employed to determine the correct dosage amount and interval.
• Embodiments of the present invention also contemplate a method of treating cancer in companion animals comprising administering a pharmaceutical composition comprising one or more of the salts of Compound I or the polymorphs of such salts of the present invention and a pharmaceutically acceptable carrier or excipient.
• the salts of Compound I or the polymorphs of such salts, as described herein would be metabolized by enzymes in the body of an organism such as a companion animal or a human being to generate a metabolite that can modulate the activity of the protein kinases. Such metabolites are within the scope of the present invention.
• Compounds of the invention may be administered alone or in combination with one or more other compounds of the invention or in combination with one or more other drugs (or as any combination thereof). It is also contemplated that the salts of Compound I or the polymorphs of such salts, as described herein, might be combined with other chemotherapeutic agents for treatment of the diseases and disorders discussed above. For example, a compound of the present invention may be combined with fluorouracil alone or in further combination with leukovorin or other alkylating agents. A compound of the present invention may be used in combination with other antimetabolite chemotherapeutic agents such as, without limitation, folic acid analogs or the purine analogs.
• a compound may also be used in combination with natural product based chemotherapeutic agents, antibiotic chemotherapeutic agents, enzymatic chemotherapeutic agents, platinum coordination complexes, and hormone and hormone antagonist. It is also contemplated that a compound of the present invention could be used in combination with mitoxantrone or paclitaxel for the treatment of solid tumor cancers or leukemias.
• Example 1 Synthesis of Compound I, ie. 5-(5-Fluoro-2-oxo-l,2-dihydro-indol-3- ylidenemethyl ' )-2,4-dimethyl- 1 H-pyrrole-3 -carboxylic acid ( 2-pyrrolidin- 1 -yl-ethyl)- amide
• 5-Fluoro-l,3-dihydro- indol-2-one was condensed with 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2-pyrrolidin-l-yl-ethyl)-amide to give Compound I.
• DMSG Dynamic Moisture Sorption Gravimetry
• hydrochloride, fumarate, and ascorbate salts were very hygroscopic (see Figure 1).
• the other two salts (citrate and phosphate) had lower moisture sorption profiles, absorbing less than 3 % water at 70% relative humidity.
• Lot 35282-CS-51 was named polymorph Form I of Compound I Phosphate. It has high crystallinity, good flowability, and large crystal size. Both the absence of the melting event at the melting temperature of Compound I free base (free base polymorph Form A, 256 0 C; free base polymorph Form B, 259 0 C) and the presence of high melting points (281 - 297 0 C) of the solids suggested that the crystals of Lot 35282-CS-51 are a different salt form and not Compound I free base. The purity of the lot was 99.6% by HPLC.
• Example 4C Estimation of Solubility of Compound I Phosphate.
• Study Design This study was a proof of target modulation study in dogs with recurrent or metastatic grade II/III MCTs. Patients received a single oral dose of Compound I phosphate at 3.25 mg/kg. Using a 6-mm punch biopsy instrument, samples were obtained from the tumor before Compound I phosphate administration and 8 hours (h) after treatment. When possible, multiple biopsies were taken. Each sample was flash frozen in liquid nitrogen and stored at -70° centigrade (C) before analysis. Blood samples for analysis of plasma levels of Compound I phosphate were obtained at the same time as tumor biopsies (see below).
• cDNA was then generated from the RNA using dNTPs, random primers, 5X First Strand Buffer, 0.1 M DTT, and Superscript Taq polymerase (all from Promega, Madison, WI). The cDNA was quantified for each sample. For the remaining samples, genomic DNA was prepared as described previously (Downing, S., Chien, M. B., Kass, P. H., Moore, P. F., and London, C. A. Prevalence and importance of internal tandem duplications in exons 11 and 12 of c-kit in mast cell tumors of dogs. Am. J. Vet. Res., 63: 1718-1723, 2002; which is incorporated by reference in its entirety).

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